Use of a tetraphenylborate (tpb) salt for the separation of biomolecules

ABSTRACT

Process for the separation of a biomolecule containing at least one cationic group from a liquid medium containing said biomolecule, which comprises the use of a tetraphenylborate (TPB) salt.

The present invention relates to a process for the separation of abiomolecule containing at least one cationic group and to a process forthe manufacture of a biomolecule involving this use.

The process of the present invention can for instance be used for themanufacture of Eptifibatide (SEQ ID NO: 5) that selectively blocks theplatelet glycoprotein IIb/IIIa receptor. It reversibly binds toplatelets and has a short half-life. It has demonstrated efficacy as anintravenous solution in the treatment of patients during coronaryangioplasty, myocardial infarction and angina.

The process of the present invention can also be used for themanufacture of Omiganan. Omiganan is the Common internationalDenomination (CTD) for the peptideH-Ile-Leu-Arg-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Lys-NH2 (SEQ ID NO 1) thatis mostly used in the form of its pentahydrochloride (5 HCl) salt.Omiganan is a cationic antimicrobial peptide. Recent research has alsoshown that it may play a role in the inflammatory response. Omiganan, inin vitro assay, demonstrated a rapid bactericidal activity againstmicro-organisms that colonize the skin and that may play a role in thepathogenesis of inflammatory diseases.

U.S. Pat. No. 5,262,567 discloses the use of a compound including aguanidine group and an unsubstituted tetraphenylborate ion asintermediate in the synthesis of peptides. As an example, the synthesisof the compound formed by arginine and tetraphenylborate (TPB) and itsuse in the synthesis of the peptide Boc-Leu-Arg-OH has been described.

It has now been found, surprisingly, that an improved separation of abiomolecule from a liquid medium, in particular a biomolecule containingat least one cationic group by the use of a tetraphenylborate (TPB) saltenables the desired biomolecule to be obtained in high yield and withhigh purity.

The invention thus relates to a process for the separation of abiomolecule containing at least one cationic group from a liquid mediumcontaining said biomolecule, which comprises the use of atetraphenylborate (TPB) salt.

For the purpose of the present invention, the term “the cationic group”refers to a functional group of the biomolecule having a positivecharge. The cationic groups in this invention are often selected from aguanidine group or an amino group. When the cationic group is an aminogroup, it is often selected from protonated primary, secondary and,preferably, tertiary amino groups. More preferably, the amino group is aquarternary (ammonium) group, for example a tetraalkylammonium group. Aguadinine group is another preferred cationic group.

For the purpose of the present invention, the term “TPB” denotestetraphenylborate. The TPB anion can be substituted on the benzene ringsor it can be unsubstituted. Preferably, the TPB anion is unsubstituted.

Examples of suitable substituted TPB anions include, for example, thetetrakis[3,5-bis(trifluoromethylphenyl]borate.

In the present invention, the TPB salt used is generally capable offorming an aqueous solution. The cation in the TPB salt is often aninorganic cation. Suitable inorganic ions are for example the sodium(Na+) and lithium (Li+). Preferred are LiTPB and NaTPB. The mostpreferably used tetraphenylborate salt is NaTPB.

In the process according to the invention, the quantity oftetraphenylborate salt used is generally from 1 to 10 equivalents oftetraphenylborate salt per cationic group present in the biomolecule,preferably this quantity is from 1 to 2 equivalents, more preferablyfrom 1 to 1.1 equivalents.

In the process according to the invention, the TBP salt is preferablyused together with an alkaline agent. The alkaline agent may be selectedin particular from inorganic bases, for instance NaHCO₃ and Na₂CO₃. Ingeneral, the selection of the alkaline agent will depend on thesensitivity of the biomolecule to be purified towards alkalineconditions.

For the purpose of the present invention, the term “biomolecule-TPBsalt” refers to biomolecule tetraphenylborate (TPB) salt.

For the purpose of the present invention, “a liquid medium containingthe biomolecule” is understood to denote in particular a solution of thebiomolecule in a solvent selected from an aqueous solvent (“aqueoussolution”), an organic solvent (“organic solution”) or mixtures thereof.

For the purpose of the present invention, the term “aqueous solvent”refers on particular to water and solutions in water of water solublecompounds, for example salt water or any other aqueous mineral saltsolution.

“Compound not miscible with water” is understood to denote in particulara compound which when contacted with an aqueous solution allows separatean aqueous phase from an organic phase by decantation.

In the present invention, the biomolecule is generally selected from thegroup consisting of a peptide, a peptide derivative, an oligonucleotidean oligonucleotide derivative and a polysaccharide.

For the purpose of the present invention, the term “peptide” refers to apolymer in which the monomers are amino acids covalently attachedtogether through amide bonds. Peptides are two or often more amino acidsmonomers long. In addition, all specific peptide sequences herein arerepresented by formulae whose left to right orientation is in theconventional direction of amino-terminus to carboxy-terminus.

For the purpose of the present invention, the term “amino acid” isintended to denote any compound comprising at least one NR₁R₂ group,preferably NH₂ group, and at least one carboxyl group. The amino acidsof this invention can be naturally occurring or synthetic. The naturalamino acids, with exception of glycine, contain a chiral carbon atom.Unless otherwise specifically indicated, the compounds containingnatural amino acids with the L-configuration are preferred. Amino acidresidues are abbreviated as follows throughout the application: Arginineis Arg or R; Lysine is Lys or K; Proline is Pro or P; Tryptophane is Tipor W, Aspartic acid is Asp or D; Cysteine is Cys or C.

For the purpose of the present invention, the term “carboxy-terminus” ofa peptide is the end of an amino acid sequence terminated by a freecarboxyl group (—COOH). On the other hand, the term “amino-terminus” ofa peptide refers to the end of an amino acid sequence terminated by anamino acid with a free amino group (—NH₂).

As used herein, the term “peptide derivative” includes an analog inwhich one or more amino acid residues have been replaced by thecorresponding D-isomer or by a non-natural amino acid residue, or achemical derivative thereof. A chemical derivative of a peptideincludes, but is not limited to, a derivative containing additionally atleast 1 chemical moiety not normally part of a peptide. Examples of suchderivatives are: (a) N-acyl derivatives of the amino terminal or ofanother free amino group, wherein the acyl group may be either analkanoyl group such as acetyl, hexanoyl, octanoyl; an aroyl group, e.g.,benzoyl, or biotinyl; (b) esters of the terminal carboxyl group or ofanother free carboxyl or hydroxy groups; and (c) amides of the terminalcarboxyl group or of another free carboxyl groups produced by reactionwith ammonia or with a suitable amine.

The term “oligonucleotide”, in the frame of the present invention,denotes in particular an oligomer of nucleoside monomeric unitscomprising sugar units connected to nucleobases, said nucleosidemonomeric units being connected by internucleotide bonds. An“internucleotide bond” refers in particular to a chemical linkagebetween two nucleoside moieties, such as the phosphodiester linkagetypically present in nucleic acids found in nature, or other linkagestypically present in synthetic nucleic acids and nucleic acid analogues.Such internucleotide bond may for example include a phospho or phosphitegroup, and may include linkages where one or more oxygen atoms of thephospho or phosphite group are either modified with a substituent orreplaced with another atom, e.g., a sulfur atom, or the nitrogen atom ofa mono- or di-alkyl amino group. Typical internucleotide bonds arediesters of phosphoric acid or its derivatives, for example phosphates,thiophosphates, dithiophosphates, phosphoramidates andthiophosphoramidates. In the present invention, the internucleotidebonds are generally protected by a suitable protective group. Aβ-cyanoethyl Group is an example of a suitable protective group.

The term “nucleoside” is understood to denote in particular a compoundconsisting of a nucleobase connected to a sugar. Sugars include, but arenot limited to, furanose rings such as ribose and 2′-deoxyribose andnon-furanose rings such as cyclohexenyl, anhydrohexitol and morpholino.The modifications, substitutions and positions indicated hereinafter ofthe sugar included in the nucleoside are discussed with reference to afuranose ring, but the same modifications and positions also apply toanalogous positions of other sugar rings. The sugar may be additionallymodified. As non limitating examples of the modifications of the sugarmention can be notably made of modifications at e.g. the 2′-or3′-position, in particular the 2′-position of a furanosyl sugar ringincluding for instance hydrogen; hydroxy; alkoxy such as methoxy,ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy,phenoxy; azido; amino; alkylamino; fluoro; chloro and bromo; 2′-4′- and3′-4′-linked furanosyl sugar ring modifications, modifications in thefuranosyl sugar ring including for instance substitutions for the ring4′-O by S, CH₂, NR, CHF or CF₂.

The term “nucleobase” is understood to denote in particular anitrogen-containing heterocyclic moiety capable of pairing with a, inparticular complementary, nucleobase or nucleobase analog. Typicalnucleobases are the naturally occurring nucleobases including the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U), and modified nucleobases including othersynthetic and naturally nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halour-acil and -cytosine,5-propynyl-uracil and -cytosine and other alkynyl derivatives ofpyrimidine bases, 6-aza-uracil, -cytosine and -thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, and fluorinatedbases. Further modified nucleobases include tricyclic pyrimidines suchas phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one),G-clamps such as a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indo1-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Other potentiallysuitable bases include universal bases, hydrophobic bases, promiscuousbases and size-expanded bases.

For the purpose of the present invention, the term “oligonucleotidederivative” refers to a class of molecules such as ‘peptide nucleicacids” (PNAs), morpholino phosphorodiamidate, peptide-oligonucleotideconjugates. The process according to the invention can suitably beapplied to cationic PNAs.

Another class of biomolecules to which the process according to theinvention can be applied is cationic polysaccharides such as cationiccellulose or cationic starch or cationic cyclodextrines. Cationicpolysaccharides are for example polysaccharides functionalized with atleast one cationic amino group as described above, in particularfunctionalized with at least one quarternary (ammonium) group.

Typical examples of peptides containing a guanidine group or an aminogroup are peptides which contain at least one amino acid selected fromarginine, homoarginine and lysine. Peptides which contain at least oneamino acid selected from arginine, and homoarginine are preferred.

Typical examples of oligonucleotides containing a guanidine group or anamino group are oligonucleotides which contain at least one nucleosideselected from adenosine, guanosine and cytosine and their derivativesthereof as listed above.

The process according to the present invention generally comprises theaddition of a TBP salt to a liquid medium containing the biomolecule.The TBP salt can be supplied in solid form, or preferably as a solution,for example an aqueous solution. Addition of TBP salt to the liquidmedium containing the biomolecule generally results in formation ofbiomolecule TPB salt.

In the process according to the invention the number of cationic groupsin the biomolecule is at least 1. Particular examples of biomoleculeswhich can be separated according to the process according to theinvention contain from 2 to 20, often from 3 to 15 cationic groups.

In the process according to the invention, the biomolecule separatedfrom the liquid medium is generally obtained in the form of a solidbiomolecule TPB salt or in the form of an organic solution suitable foruse in a further reaction step.

When a solid biomolecule TPB salt is desired, the process according tothe invention generally comprises precipitating or crystallizing thesolid biomolecule TPB salt from a liquid medium containing thebiomolecule. In this case, the liquid phase can be suitably contactedwith an aqueous solution. Such aqueous solution may be, for example,water, salt water or any other aqueous mineral salt solution. In thiscase it is particularly advantageous to control the pH of the aqueoussolution. The pH value of the aqueous solution is preferably controlledto be less than or equal to 10 more preferably, less than or equal to 9,still more preferably, less than or equal to 7.5. The pH value of theaqueous solution is preferably controlled to be higher than or equal to5, more preferably, higher than or equal to 6.0, most preferably, higherthan or equal to 6.5. The pH value of the aqueous solution may becontrolled by the addition of mineral salts. Suitable salts which can beused include alkali or earth alkali chlorides, in particular sodiumchloride, alkali or earth alkali sulphates, in particular potassiumsulphate, alkali or earth alkali hydrogen carbonates, in particularsodium hydrogen carbonate. Particular preferred are salt water solutionsincluding sodium chloride, preferably in an amount of 5% by weight ofthe salt water solution.

The precipitation or crystallization is generally carried out at atemperature from 0° C. to 20° C., preferably from 0° C. to 5° C.

The precipitated or crystallized biomolecule-TPB salt can be separatedfrom the aqueous solution for example by filtration, decantation,centrifugation or spray drying.

According to one suitable approach, the biomolecule-TPB salt iscollected via filtering and optionally washed. The biomolecule-TPB saltis preferably dried before optionally being submitted to furtherprocessing steps such as further reaction steps, lyophilisation,packaging and/or storage.

The invention will now be further explained with regard to specificembodiments.

In a first specific embodiment of the present invention, the liquidmedium is an aqueous solution.

In this first aspect of the present invention, the aqueous solution ofthe biomolecule containing at least one cationic group can be obtained,for example, by adding an aqueous solution to the synthesis solution forthe manufacture of said biomolecule in an organic solvent or solventmixture.

Said aqueous solution containing the biomolecule containing at least onecationic group is then generally washed with an organic washing solvent.The organic washing solvent is preferably chosen from compounds whichare not miscible with water. Non limiting examples of suitable organicwashing solvents are selected from halocarbons such as dichloromethane(DCM) and chloroform, esters such as ethyl acetate (AcOEt) and isopropylacetate (AcOiPr) and ethers such as diethyl ether, diisopropyl ether andmethyl-tert.butylether (MTBE).

The optional washing step allows removing together with the organicphase organic reaction solvents used in the reaction medium, for exampleN,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF),N-methylpyrrolidone (NMP), reactants and/or starting reagents.

In a first aspect of the first embodiment of the present invention, thebiomolecule containing at least one cationic group present in theaqueous solution is precipitated or crystallized, as described above,from the aqueous solution as a solid biomolecule tetraphenylborate (TPB)salt [biomolecule-TPB salt]. The precipitation or crystallisation can berealized, for example, by pouring the aqueous solution containing saidbiomolecule into an aqueous solution containing the TPB salt. In anotherexample, an aqueous solution containing the TPB salt is added to theaqueous solution containing the biomolecule. The precipitated orcrystallised biomolecule TPB salt can be further processed, as describedabove.

In a second aspect of the first embodiment of the process according tothe invention, the biomolecule containing at least one cationic grouppresent in the aqueous solution is extracted as a biomolecule-TPB saltinto a first organic solution. The extraction is in general performed bycontacting the aqueous solution with the TPB salt and a suitable organicsolvent or solvent mixture. Preferred organic solvents are selected fromhalocarbons such as dichloromethane (DCM) and chloroform, esters such asethyl acetate (AcOEt) and isopropyl acetate (AcOiPr) and ethers such as,diethyl ether, diisopropyl ether and MTBE, alone or in combination withalcohols such as for example n- butanol, iso-butanol and sec-butanol.Good results are obtained using dichloromethane.

In this aspect of the first embodiment of the process according to theinvention, it is particularly advantageous to control the pH of theaqueous solution containing the biomolecule. The pH value of thisaqueous solution is preferably controlled to be less than or equal to10; more preferably, less than or equal to 9, still more preferably,less than or equal to 7.5. The pH value of this aqueous solution ispreferably controlled to be higher than or equal to 5, more preferably,higher than or equal to 6.0, most preferably, higher than or equal to6.5. The pH value of this aqueous solution may be controlled, forexample, by the addition of an aqueous mineral salt solution. Suitablesalts to be used in the above mentioned salt water solutions includealkali or earth alkali chlorides, in particular sodium chloride, alkalior earth alkali sulphates, in particular potassium sulphate, alkali orearth alkali hydrogen carbonates, in particular sodium hydrogencarbonate.

In a third aspect of the first embodiment of the process according tothe invention, the invention relates to a process for the manufacture ofa biomolecule-TPB salt containing at least one cationic group whichcomprises

-   -   (a) providing an aqueous solution of the biomolecule containing        at least one cationic group    -   (b) adding a TPB salt to the aqueous solution    -   (c) recovering the biomolecule-TPB salt from the aqueous        solution.

In a second embodiment of the process according to the invention, theliquid medium is an organic solution.

In this second aspect, the liquid medium is an organic solution to whichafter optional washing with an aqueous solution, the tetraphenylboratesalt is added thereby providing a first organic solution containing thebiomolecule-TPB salt. The organic solution of the biomolecule containingat least one cationic group can be obtained, for example, by diluting asynthesis solution for the manufacture of a biomolecule in an organicsolvent or solvent mixture, in particular a reaction solvent asdescribed above, after the completion of a synthesis step with anorganic dilution solvent. The organic dilution solvent is preferablychosen from compounds which are not miscible with water. Preferably,organic dilution solvents are selected from halocarbons such asdichloromethane (DCM) and chloroform, esters such as ethyl acetate(AcOEt) and isopropyl acetate (AcOiPr) and ethers such as diethyl ether,diisopropyl ether and MTBE, alone or in combination with alcohols suchas for example n-butanol, iso-butanol, sec-butanol. Good results areobtained using dichloromethane.

Said organic solution of the biomolecule containing at least onecationic group can be optionally washed with an aqueous solution, asdescribed above, and further washed with an aqueous solution containinga TPB salt thereby providing a first organic solution in which thebiomolecule remains dissolved as a biomolecule-TPB salt. The differentwashing steps allow to reduce impurities and other unwanted compoundscontent in the first organic solution.

In a first advantageous aspect of the second embodiment of processaccording to the invention, the first organic solution obtained issubjected to a further reaction step without isolation of thebiomolecule-TPB salt.

A second advantageous aspect of the second embodiment of processaccording to the invention relates to a process for the manufacture of abiomolecule-TPB salt containing at least one cationic group comprisesthe following steps

-   -   (a) providing an organic solution of the biomolecule containing        at least one cationic group    -   (b) adding a TPB salt to the organic solution    -   (c) recovering the biomolecule-TPB salt from the first organic        solution.

In a third embodiment of the process according to the invention, thefirst organic solution containing the biomolecule-TPB salt which can beobtained, for example, as described in the first and second embodimentabove, can be treated with a further organic solvent or solvent mixtureto obtain a second organic solution containing the biomolecule-TPB salt.The further organic solvent or solvent mixture may be added directly tothe first organic solution containing the biomolecule-TPB salt or may beadded after optional further treatment of said first organic solution.In this latter case which is preferred, the first organic solutioncontaining the biomolecule-TPB salt may for example be concentrated, inparticular by evaporation under vacuum, before the addition of thefurther organic solvent or solvent mixture. The further organic solventpreferably comprises at least one component selected from alcohols suchas, preferably, methanol or methoxyethanol, amide type solvents such asN,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF),N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), halocarbons such asdichloromethane (DCM) and chloroform, esters such as ethyl acetate(AcOEt) and isopropyl acetate (AcOiPr) and ethers such as diethyl ether,diisopropyl ether, dioxane, tetrahydrofuran (THF) and MTBE; pyridine,acetonitrile, or mixtures thereof. More preferably, the further organicsolvent is selected from methanol, methoxyethanol, N,N-dimethylacetamide(DMA), N-methylpyrrolidone (NMP) and N,N-dimethylformamide (DMF). Goodresults are obtained with methanol or methoxyethanol.

In a first advantageous aspect of the third embodiment of the processaccording to the invention, the second organic solution obtained issubjected to a further reaction step without isolation of thebiomolecule-TPB salt.

In a second advantageous aspect of the third embodiment of the processaccording to the invention, the biomolecule-TPB salt is separated fromthe second organic solution as a solid biomolecule-TPB salt byprecipitation or crystallisation, as described above. The precipitationor crystallisation can be performed by the addition of the secondorganic solution containing the biomolecule-TPB salt to an aqueoussolution. Alternatively, the precipitation or crystallisation can alsobe performed by the addition of an aqueous solution to said secondorganic solution. The precipitated or crystallised biomolecule TPB saltcan be further processed as described above.

In a third advantageous aspect of the third embodiment of the processaccording to the invention, the second organic solution containing thebiomolecule-TPB salt is further subjected to a concentrating step. Theconcentrating step is often carried out by evaporation under vacuum.

In a forth embodiment of the process according to the invention, thefirst organic solution containing the biomolecule-TPB salt, which can beobtained, for example, as described in the first or second embodimentabove, is subjected to a concentrating step, for example as describedhere before, to provide a concentrated organic solution containing thebiomolecule-TPB salt.

The biomolecule-TPB salt may be separated from the concentrated organicsolution, which can be obtained as described in the third and forthembodiments, for example, by precipitation or crystallisation, asdescribed above. The precipitation or crystallisation can be performedby pouring the concentrated organic solution containing thebiomolecule-TPB salt into an aqueous solution. Alternatively, theprecipitation or crystallisation can also be performed by the additionof an aqueous solution to said concentrated organic solution. Theprecipitated or crystallised biomolecule TPB salt can be furtherprocessed as described above.

The resulting concentrated organic solution containing thebiomolecule-TPB salt can be used directly for a further reaction step.Optionally, said concentrated organic solution can be diluted with afurther organic solvent as described here before and then subjected to afurther reaction step.

The different embodiments of the process according to the invention canpreferably be applied to a biomolecule which is selected from the groupconsisting of a peptide containing at least one cationic group or aderivative thereof, more particularly, peptide or peptide derivativecontaining a guanidine group or an amino group. Typical examples ofpeptides or peptide derivatives containing a guanidine group or an aminogroup are peptides which contain at least one amino acid selected fromarginine (Arg), homoarginine (homo Arg) and lysine (Lys).

The invention thus relates in another specific aspect to a process forthe manufacture of a peptide or a derivative thereof comprising one ormore amino acid units containing a cationic group, comprising the steps

-   -   (a) coupling in an organic solution a first amino acid or a        first peptide with a second amino acid or second peptide,        wherein the first and/or the second amino acid or peptide        contains a cationic group, to produce a peptide containing a        cationic group.    -   (b) adding a TPB salt after the completion of the coupling step;        and    -   (c) separating as described herein before the peptide containing        a cationic group or the derivative thereof from an aqueous        solution or an organic solution.

The process according to the invention is especially advantageous if thenumber of cationic groups in the peptide is from 2 to 20, preferablyfrom 3 to 15.

The process of the present invention is especially beneficial when thecoupling step involves a peptide containing the amino acid Arg or Har.It has been found surprisingly that the use of a protecting group forthe side chain in Arg or Har can be omitted when a tetraphenylborate insteps (b) and (c). The peptide manufacturing process according to theinvention can be advantageously applied to the manufacture of Omiganan(SEQ ID NO: 1) or Eptifibatide (SEQ ID NO: 5).

In step (a) of the peptide manufacturing process of the presentinvention, protecting groups can be used in general for one or moreamino groups in the amino acids or peptide intermediates involved.

By way of illustration, the following protecting groups may be employedin the peptide manufacturing process of the invention: acyl-typeprotecting groups such as formyl, trifluoroacetyl, phthaloyl,4-toluenesulfonyl, benzenesulfonyl and 2-nitrophenylsulfenyl, aromaticurethane-type protecting groups such as substituted or unsubstitutedbenzyloxycarbonyl (Z), p-bromobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, benzhydryloxycarbonyl, 2-(4-biphenylyl)prop-2-yl-oxycarbonyl, dimethyloxphenyl) prop-2-yl-oxycarbonyl, andtriphenylphosphonoethyloxycarbonyl, aliphatic urethane-type protectinggroups, in particular tert-butyloxycarbonyl (BOC),diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc),2-methylsulfonylethyloxycarbonyl and 2,2,3-trichloroethyloxycarbonyl,cycloalkyl urethane-type protecting groups such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl,tert-amyloxycarbonyl and isobornyloxycarbonyl, thiourethane-typeprotecting groups, in particular phenylthiocarbonyl, alkyl-typeprotecting groups such as especially triphenylmethyl (trityl) and benzyltrialkylsilane groups such as, for example tert.butyl-dimethylsilyl andalkoxy groups such as for example methyl ester, ethyl ester, tert-butylester and benzyl ester and a p-nitrobenzylester.

In the peptide manufacturing process of the invention, the first aminoacid or peptide has in general an activated carboxyl group. Variousactivating groups may be used in the process of the invention, forexample groups derived from N,N-dicyclohexycarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), pivaloyl chloride(PivCl), i-butylchloroformate (IBCF). In addition to these activatinggroups additives are sometimes used advantageously. Preferred additivesare N-hydroxysuccinimide (Suc-OH), N-hydroxybenzotriazole (HOBt), or3,4-dihydro-3-hydroxy-4-oxo-1,2,3 benzotriazine (HOOBt).

A further particular aspect of the peptide manufacturing processaccording to the invention is related to a process in which the aqueousor organic solution of a peptide containing at least one cationic groupor derivative thereof is obtained by adding an aqueous solution to thesynthesis solution for the manufacture of said peptide in an organicsolvent after the completion of a coupling step.

In general, the synthesis solution after a peptide coupling step may ormay not contain a base. In consequence, the subsequent steps may dependon whether or not a base is present.

In a first embodiment of the peptide manufacturing process according tothe invention where no base is present in the synthesis solution, it ispreferable to dilute the synthesis solution with an aqueous solution ofan acid for example hydrochloric acid. A preferably, the solution isthen washed with an organic solvent or mixtures thereof. The organicsolvent is preferably chosen from compounds which are not miscible withwater. Non limiting examples of such suitable organic solvents areselected from halocarbons such as dichloromethane (DCM) and chloroform;esters such as ethyl acetate (AcOEt) and isopropyl acetate (AcOiPr) andethers such as diethyl ether, diisopropyl ether and MTBE. A number ofdifferent solvents may be used. Especially advantageous is the use ofdichloromethane, ethyl acetate, isopropyl acetate, diisopropyl ether,and MTBE, alone or in combination. The washing step allows removingtogether with the organic phase organic solvents used in the reactionmedium (for example DMF, DMA, NMP), reactants (for example HOBt, HOOBt)and/or starting amino acids or peptides.

In this embodiment of the present invention, the peptide containing atleast one cationic group or derivative thereof is present in the aqueoussolution and the addition of a TPB salt to the aqueous solution and therecovery of the tetraphenylborate (TPB) salt of the peptide orderivative thereof can be carried out according to the process accordingto the invention, as described above, in particular as described in thefirst embodiment of the process according to the invention.

In a second embodiment of the peptide manufacturing process according tothe invention wherein a base is present in the synthesis solution, it ispreferred to dilute a synthesis solution with a dilution solvent asdescribed above. In a second step, the organic solution comprisingorganic solvents (organic phase) may be advantageously washed to theaqueous solution of an acid. This washing step may allow the eliminationthrough the aqueous phase of starting compounds, especially when theycontain Arg, as well as certain basic additives (for example TEA, DIPEA)and reactants (for example EDU) which might form a salt with TPB.

In this embodiment of the invention, the peptide containing at least onecationic group or derivative thereof is present in the organic solutionand the addition of a TPB salt to the organic solution and the recoveryof the tetraphenylborate (TPB) salt of the peptide a derivative thereofcan be realized according to the process of the present invention, asdescribed in the different embodiments above.

In one particular embodiment, which is particularly suitable in theframework of synthesis of Omiganan (SEQ ID NO: 1), the peptide synthesisprocess according to the invention comprises forming a tetraphenylboratesalt of an Arg containing peptide. Typically said tetraphenylborate saltof an Arg containing peptide is formed by contacting a coupling stepreaction medium containing an Arg containing peptide, which is usuallyobtained by a coupling step according to the process according to theinvention with a source of tetraphenylborate anions. Tetraphenylboratesalts are suitable as source of tetraphenylborate anions.

Hence, it is preferred to perform at least one step in the presence of atetraphenylborate salt (TPB) which is preferably added after thecompletion of at least one coupling step.

The cation in the tetraphenylborate (TPB) salt can be inorganic ororganic. Examples of organic ions are the tetraethylammonium,diisopropylethylammonium, N-ethylpiperidinium, N-methylmorpholinium,N-ethylmorpholinium. Suitable inorganic ions are for example the sodium(Na⁺) or lithium (Li⁺). Most preferably, a tetraphenylborate salt isused that is capable of forming an aqueous solution. Preferred are LiTPBand NaTPB. The most preferably used tetraphenylborate salt is NaTPB.

The TPB anion can be substituted on its benzene ring or it can be usedwithout any substitution. Preferably, the TPB anion is not substituted.Examples of suitable substituted TPB anions include, for example, thetetrakis(3,5-bistrifluoromethylphenyl)borate.

The quantity of the tetraphenylborate salt employed may vary within widelimits. Preferably, from 1 to 10 equivalents, preferably 1 to 1.5equivalents of tetraphenylborate salt is employed per Arg unit in theArg containing peptide.

The tetraphenylborate salt is preferably employed in the process of thepresent invention during the work-up after the completion of at leastone coupling step, in the presence of a solvent or a mixture ofsolvents. Suitable solvents are especially methanol, methoxyethanol,dichloromethane, n-butanol, iso-butanol, sec-butanol, and tent-butanol.Good results were obtained using methoxyethanol.

In this embodiment, said tetraphenylborate salt of an Arg containingpeptide can advantageously be subjected without isolation to at leastone further synthesis step. Often, said tetraphenylborate salt of an Argcontaining peptide is subjected at least to a deprotection step followedby a coupling step.

The invention also relates to a solid biomolecule-TPB salt containing atleast one cationic group. Peptides, in particular as herein describedand more particularly Arg or Har containing peptides are preferredbiomolecules in the solid biomolecule-TPB salt according to theinvention may notably be obtained according to the process of theinvention.

It has been found, surprisingly, that the solid biomolecule-TPB salt issubstantially stable when stored.

“Substantially stable” is understood to denote in particular the factthat when comparing the biomolecule content of the biomolecule-TPB saltbefore and after storage, the biomolecule-TPB salt content found afterstorage is at least 98%, preferably at least 99% of the biomoleculecontent before storage.

The invention consequently also relates to a method of storing the solidbiomolecule-TPB salt according to the invention

The storage of the solid biomolecule-TPB salt is generally carried outat a temperature less than or equal to 25° C.; more preferably, lessthan or equal to 22° C., still more preferably, less than or equal to20° C. The storage of the solid biomolecule-TPB salt is generallycarried out at a temperature higher than or equal to −90° C., oftenhigher than or equal to −80° C. Preferably, the storage is carried outat a temperature higher than or equal to −20° C., more preferably higherthan or equal to 0° C.

It has been found that use of the solid biomolecule-TPB salt accordingto the invention allows for long storage with relatively low energyconsumption

The solid biomolecule-TPB salt according to the invention is oftenstored for a time period longer than 24 hours, for example longer than 1week. Stability may be observed for periods of time as long as 1 year,or longer. Often the storage time period will not exceed 6 months.

The invention also relates to the use of solid biomolecule-TPB salts assource of intermediate in biomolecule synthesis. The use according tothe invention applies preferably to peptide fragments to be used in acoupling, deprotection or purification step.

The invention relates also to a process for the manufacture of a salt ofa biomolecule containing at least one cationic group which comprises (a)providing a solution of a TPB salt of the biomolecule (b) contactingsaid solution with a counter-ion salt, the cation of which forms a saltwith tetraphenylborate which is less soluble in the solution than theTPB salt of the biomolecule, so as to exchange the tetraphenylborateanion against the counter-ion (c) optionally recovering the counter-ionsalt of the biomolecule from the solution.

In one embodiment, the ion exchange may be realized by adding to thesolution a counter-ion salt, the cation of which forms a salt with thetetraphenylborate ion. Generally, the cation and solvent is chosen suchthat upon contacting the solution with the counter-ion salt, the cationTPB salt will precipitate from the solution. Suitable counter-ion saltsare generally selected from potassium salts, for example, potassiumchloride and potassium acetate and, preferably quaternary ammoniumsalts, for example tetraalkyl ammonium acetate and ammonium chloride. Atetraalkyl ammonium chloride, such as benzyltrimethylammonium chloridegives good results. In this specific embodiment of the presentinvention, the biomolecule-TPB salt is generally dissolved in an organicsolvent. The organic solvent is preferably chosen from alcohols such asmethanol or ethanol. Methanol is the most preferred alcohol. Acombination of a cation chosen from quaternary ammonium cations forexample benzyltrimethylammonium cation and an alcohol is particularlypreferred.

The molar ratio counter-ion salt/TPB anion used in this embodiment isgenerally about 1, preferably from 1 to 1.1.

In another embodiment, the ion exchange is carried out by using an ionexchange resin. In this embodiment the exchange is preferably performedby passing the biomolecule-TPB salt solution through a column containingthe exchange resin. Suitable examples of exchange resins containimmobilized amino cationic groups as described above, in particularquaternary (ammonium), groups with the desired counter-ion, inparticular acetate or chloride. A specific example of suitable ionexchange resin is commercialized by Rohm and Haas under the denominationIRA 958. The exchange resin can be optionally washed with an organicsolvent preferably chosen from alcohols such as methanol, ethanol.Methanol is most preferred alcohol.

The following examples are intended to illustrate the invention without,however, limiting its scope.

The ratios indicated refer to volume ratios. If nothing else isindicated, the purity of the compound was more than 98% by weight andthe ratios indicated refer to volume ratios. In cases where iso-butanolwas used, sec-butanol could be used as well.

In these examples and throughout this specification the abbreviationsemployed are defined as follows: Boc is t-butoxycarbonyl, n-BuOH isn-butanol, DCM is dichloromethane, DIPEA is N,N-diisopropylethylamine,DMF is N,N-dimethylformamide, DMA is N,N-dimethylacetamide, Fmoc isfluorenylmethyloxycarbonyl, HBTU isN,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium-hexafluororphosphate), HOBt is 1-hydroxybenzotriazole, HOOBT is3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, EDC isN,N-dimethylaminopropylcarbodiimide, DCC is dicyclohexyl carbodiimide,i-BuOH is iso-butanol, IPE is diisopropylether, McCN is acetonitrile,McOH is methanol, NMM is N-methylmorpholine, NMP is1-methyl-2-pyrrolidone, THF is tetrahydrofuran, pNA is p-nitroanilinamide, Tos is tosyl, MTBE is methyl-tert-butylether.

EXAMPLE 1 Synthesis of HCLH-Arg-Lys(Boc)-NH₂

1.02 equivalents of Z-Arg-OH.HCl (Mw=344.8) were added at roomtemperature to a mixture of DMA and CH₂Cl₂ (6/4). Thereafter, 1.03equivalents of HOBt (N-Hydroxybenzotriazole, Mw=135.12) and 1.00equivalent of H-Lys(Boc)-NH₂ (Mw=245.3; purity: 99%) were added. Aftercooling the solution to 0±5° C., 1.03 equivalents of EDC.HCl(Mw 191.7)were added.

Stirring was continued for further 30 min at 0±5° C. and then for atleast 2 hours at room temperature. After checking for the completion ofthe reaction by HPLC, the reaction mixture was diluted with aCH₂Cl₂/iso-BuOH (6/4) mixture and extracted with a solution of 0.5 eq.of HCl. The acidic aqueous phase was extracted a second time with amixture of CH₂Cl₂/iso-BuOH (6/4).

The combined organic phases were first washed with a 5% (weight) aqueoussolution of Na₂CO₃ containing 1.05 equivalents of sodiumtetraphenylborate (TPBNa) (Mw=342 g,), and then 4 times with a 5%(weight) aqueous NaCl solution.

After the organic phase had been concentrated in vacuo, methoxyethanolwas added in several portions to the concentrate to eliminate traces ofiso-butanol. It was then further evaporated. The concentrate was thenfinally diluted with methoxyethanol and slowly added to a cold (0 to 5°C.) 5% (weight) aqueous NaCl solution. The peptide precipitated and waskept for at least 30 min at low temperature and then filtered.

The solid was washed 3 times with cold (0±5° C.) demineralised water.Thereafter, the solid was redissolved in MeOH and stirred until aslightly cloudy solution was obtained. The solution was partiallyconcentrated and the methanolic solution was then added slowly to acooled aqueous NaCl 5% (weight) solution. The precipitate was kept forat least 30 min at low temperature before it was filtered off. Finallythe solid was washed 3 times with cold demineralised water (0° C.±5° C.)and dried under vacuum (45° C.). An off white solid was finallyobtained. The yield based on the NMR measurement of the content was 89%.

Synthesis of HCl.H-Arg-Lys(Boc)-NH₂

A methanol solution of 1.00 equivalent of TPB.Z-Arg-Lys(Boc)-NH₂(Mw=535.6; purity=62.0%) was passed several times through a columncontaining a methanol washed resin IRA 958 (Mw=1000; 3.00 equivalents).After checking the exchange by HPLC, the resin was filtered and washedthree times with methanol. The combined organic phases were partiallyconcentrated in vacuo. The concentrated solution was diluted with water.

Pd catalyst (Mw=106.4; 2% weight) were added and the suspension thenhydrogenated for at least 5 hours at 35±5° C. The catalyst was filteredoff, washed twice with a mixture methanol/water. The filtrate wasevaporated in vacuo, the residue suspended in DMA and partiallyevaporated in vacuo in order to eliminate traces of water. Afterchecking the water content, the final solution was titrated by HCl(0.1N) and further used without any purification.

Yield (based on the titration): 90%.

EXAMPLE 2 Synthesis of 2HCl.H-1-Trp-Arg-Arg-Lys(Boc)-NH₂(SEQ ID NO: 2)Synthesis of Z-Trp-Arg-Arg-Lys(Boc)-NH₂.2TPB (SEQ ID NO: 2)

1.00 equivalent Z-Trp-Arg-OH (Mw=494.5; purity=85.0%,) and 1.10equivalents HOOBt (Mw=163.13) were added to the DMA solution of 1.15equivalents HCl.H-Arg-Lys(Boc)-NH₂ (Mw=437.5; purity=20.0%;) which hadbeen previously diluted with CH₂Cl₂. After cooling the solution to −5±5°C., 1.00 equivalent HCl/dioxane 4N was slowly poured in and then 1.10equivalents EDC (Mw=191.7) were added. The reaction mixture was stirredat −5±5° C. for at least 3 hours and then at least for 8 hours at 5±5°C. After checking the completion of the reaction by HPLC, the reactionmixture was diluted with a CH₂Cl₂/sec-butanol mixture (6/4), washedfirst with a 5% (weight) aqueous solution of NaCl containing HCl (0.5eq.), then with 1900 ml of a 5% (weight) aqueous solution of Na₂CO₃containing 2.2 equivalents NaTPB (,Mw—342), and finally five times witha 5% (weight) aqueous solution of NaCl. After the concentration of theorganic layer, the residue was dissolved in methanol and thenconcentrated in vacuo in order to eliminate most of the remainingCH₂Cl₂. This final solution was titrated by NMR and further used withoutany purification.

Yield (based on titration content)=83%.

Synthesis of 2HCl.H-Trp-Arg-Arg-Lys(Boc)-NH₂(SEQ ID NO: 2)

The methanol solution of 1.00 equivalent of 2TPB.Z-Trp-Arg-Arg-Lys(Boc)-NH₂ (Mw=878.11) were passed several times through a columncontaining 6.00 equivalents of a methanol washed resin IRA 958(Mw=1000). After checking the exchange by HPLC, the resin was filteredand washed three times with methanol. The combined organic phases werepartially concentrated in vacuo. The concentrated solution was dilutedwith water and Pd catalyst were added. The suspension was thenhydrogenated for at least 3 hours at 35±5° C. The catalyst was filteredoff and washed twice with methanol. The filtrate was evaporated invacuo, the residue dissolved in DMA and further concentrated in order toeliminate the remaining water. After the water content was checked, theprecipitate was dissolved in DMA and partially evaporated in vacuo inorder to adapt the weight of the solution. The final solution wastitrated by 0.1N HCl and further used without any purification.

Yield (based on the titration): 95%.

EXAMPLE 3 Synthesis of 2HCl.H-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH₂ (SEQID NO: 3) Synthesis of Z-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH₂.2TPB (SEQID NO: 3)

1.00 equivalent of Z-Trp-Trp-Pro-OH (Mw=621.7; purity=94.0%) was addedto the DMA solution of 1.05 equivalents 2HCl.HTrp-Arg-Arg-Lys(Boc)-NH₂(Mw=816.9; purity=15.0%) previously diluted with CH₂Cl₂. Then, 1.20equivalents N,N′-Diisopropylethylamine (DIPEA) (Mw=129.2) and 1.05equivalents of2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate)(HBTU) (Mw=379.24) were added.

The reaction mixture was stirred at room temperature for at least 1hour. After checking the completion of the reaction by HPLC, thereaction mixture was diluted with a CH₂Cl₂/iso-butanol mixture (8/2),washed first with a 5% (weight) aqueous solution of NaCl and HCl (1.5eq.), then with a 5% (weight) aqueous solution of Na₂CO₃ and 2.2equivalents NaTPB (Mw=342 g/mol), and finally three times with a 5%(weight) aqueous solution of NaCl. After concentration of the organiclayer, the residual oil was several times dissolved in methoxyethanol,then concentrated in vacuo in order to eliminate most of the remainingiso-butanol. After GC control, the concentrate was precipitated byslowly pouring it into cold (0±5° C.) 5% (weight) aqueous solution ofNaCl. After stirring for at least 1 hour, the suspension was filteredand washed twice with cold water. The precipitate was dried in vacuo at45° C. A white solid was finally obtained.

Yield (based on NMR content)=98%.

Synthesis of 2HCl.H-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH₂(SEQ ID NO: 3)

A methanol solution of 1.00 equivalent of2TPB.Z-TrpTrp-Pro-Trp-Arg-Arg-Lys(Boc)-NH₂ (SEQ ID NO: 3) (Mw=1347.5;purity=64.0%) was passed several times through a column containing amethanol washed resin IRA 958 (or Amberjet Cl 1000; 6.00 equivalents).After checking the exchange by HPLC, the resin was filtered, washedthree times. The combined organic phases were partially concentrated invacuo and then diluted with water. Finally, 2% (weight) of Pd catalystwere added and the suspension hydrogenated for at least 3 hours at 40°C. The catalyst was filtered off, washed three times with a mixture ofmethanol/water. The combined filtrates were evaporated in vacuo, theresidue suspended in DMA and further concentrated in order to eliminatethe remaining water. After checking the water content, the solution wastitrated by HCl (0.1 N), AgNO₃ (0.1N) or NMR and further used withoutany purification.

Yield (based on titration)=82%.

EXAMPLE 4 Synthesis ofZ-Arg-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH₂.3TPB (SEQ ID NO: 4)

1.00 equivalent of Z-Arg-Trp-Pro-OH (Mw=591.65; purity=96.0%), 1.00equivalent of HCl/dioxane (4N;) and 1.05 equivalents HOBt (Mw=135.12;purity=98.0%) were added to 1.00 equivalent2HCl.H-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH₂ (SEQ ID NO: 3) (Mw=1213.4;purity=85.0%;) in solution in DMA diluted with CH₂Cl₂. After thesolution had been cooled to 10±5° C., 1.02 equivalents EDC (Mw=191.7)were added. The reaction mixture was stirred at 10±5° C. for 30 min andthen at least 4 hours at room temperature. After the completion of thereaction was confirmed by HPLC, the reaction mixture was diluted with aCH₂Cl₂/iso-butanol mixture (8/3), washed first with a 5% (weight)aqueous NaCl solution with HCl (0.5 eq.), then with a 5% (weight)aqueous solution of Na₂CO₃, containing 3 eq NaTPB (Mw=342), and finallytwice with a 5% (weight) aqueous solution of NaCl. After concentrationof the organic layer, the residual oil was several times dissolved inmethoxyethanol, then concentrated in vacuo in order to eliminate most ofthe iso-butanol. After GC control, the concentrate was precipitated byslowly pouring it into cold 5% (weight) aqueous solution of NaCl. Afterstirring for at least 1 hour, the suspension was filtered, washed twicewith cold water. The precipitate was dried at 40±5° C. An off whitesolid was finally obtained.

Yield (based on NMR content)=87%. A methanol solution of 1.00 equivalentof 3TPB.Z-Arg-Trp-Pro-TrpTrp- Pro-Trp-Arg-Arg-Lys(Boc)-NH₂ (Mw=1787.1;purity=57.0%;) was passed several times through a column containing amethanol washed resin TRA 958 (Mw=1000, 9.00 equivalents). After theexchange had been checked by HPLC, the resin was filtered and washedthree times with methanol. The combined organic phases were partiallyconcentrated in vacuo. The concentrated solution was then diluted withwater and Pd catalyst was added. The suspension was then hydrogenatedfor at least 6 hours at 35±5° C. The catalyst was filtered off, washedthree times with a mixture methanol/water. The combined filtrates wereevaporated in vacuo, the residue suspended in DMA and furtherconcentrated in order to eliminate the remaining water. After checkingthe water content, the solution was titrated by HCl (0.1N) or NMR andfurther used without any purification.

Yield (based on NMR content measurement): 93%.

EXAMPLE 5 Synthesis of Z-(D)Arg-Gly-Arg-pNA.2HCl

Synthesis of Z-(D)Arg-Gly-Arg-pNA.2HCl

7.7 g of Z-(D)ArgOH (25 mmoles) and 13.8 g of 2HCl.H-Gly-ArgpNA weredispersed in about 100 ml of DMA at room temperature till completedissolution. The mixture was then cooled to 0° C. and DIPEA(N,N′-diisopropyl ethyl amine) was added to neutralize the excess ofHCl, followed by 3.6 g of HOBt (26.13 mmoles) and 5.7 g of DCC (27.5mmoles). The solution was left to stir at least 1 hour at 0° C. beforebeing conditioned to 25±5° C. When the reaction was considered ascompleted (followed by HPLC), the crude was concentrated in vacuo, andthe concentrate was then diluted with water to precipitate the DCU whichwas then removed by filtration and washed with water. The aqueoussolution was washed several times with DCM to remove DMA (dimethylacetamide) and HOBt. Two equivalents of TPBNa and 500 ml of DCM werepoured into the aqueous solution while the pH was adjusted between 6.5and 7.5 by the controlled addition of an aqueous NaHCO3 solution. After1 hour of mixing, the aqueous phase was discarded and the organic phasewas washed several times with an aqueous solution of NaCl and finallywith water. The solvent was removed under vacuum and replaced with MeOH.

Isolation of Z-(D)Arg-Gly-Arg-pNA.2TPB,

The methanolic solution of Z-(D)Arg-Gly-Arg-pNA.2TPB was then pouredinto an aqueous 5% solution of NaCl at 0±5° C. to precipitateZ-(D)Arg-Gly-Arg-pNA.2TPB. After filtration, washing by water and dryingunder vacuum, Z-(D)Arg-Gly-Arg-pNA.2TPB was obtained as a white solid.

Isolation of the Bischlorhydrate Salt of Z-(D)Arg-Gly-Arg-pNA

The TPB salt was dissolved in methanol and the solution was passedseveral times through a column containing a methanol washed resin IRA958. After checking the exchange by HPLC, the resin was washed severaltimes with methanol. The combined concentrated filtrates wereconcentrated under vacuum and the solid obtained was purified by HPLCand finally lyophilized to yield Z- (D)Arg-Gly-Arg-pNA as itsbischlorhydrate salt.

EXAMPLE 6 Synthesis of Ac-(D)Arg-Gly-Arg-pNA.2HCl

Isolation of Purified Ac-(D)Arg-Gly-Arg-pNA.2TPB,

The pH of a Ac-(D)Arg-Gly-Arp-pNA.2TFA solution in water/acetonitrile(31 l) was adjusted to 7±0.5 by adding an aqueous solution of NaHCO₃.The acetonitrile fraction was evaporated in vacuo (the volume of thesolution was maintained by adding water if necessary). 1 kg TPBNa and1.1 kg NaHCO₃ were dissolved in about 20 1 water. The concentratedpeptide solution was then gradually poured into the aqueous solutionTPBNa/NaHCO₃. The Ac-(D)Arg-Gly-Arg-pNA.2TPB precipitated. Afterfiltration, washing by water (40 l) and drying under vacuum (45° C.),1.6 kg of Ac-(D)Arg-Gly-Arg-pNA.2TPB were obtained.

Isolation of the Purified Ac-(D)Arg-Gly-Arg-pNA.2HCl,

The TPB salt was dissolved in 20 l methanol which was passed severaltimes through a column containing a methanol washed resin IRA 958 (7.6kg resin). After checking the exchange by HPLC, the resin was washedseveral times with methanol (11 l each time). The combined concentratedfiltrates were precipitated in 44 l cooled (−5°±5° C.) acetonitrile.After washing with acetonitrile (12 l), drying under vacuum, theprecipitate gave 0.8 kg of off white solid. If necessary, theAc-(D)Arg-Gly-Arg-pNA.2HCl can be precipitated a second time.

EXAMPLE 7 Synthesis of Mpr(*)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(*)-NH₂ (SEQID NO: 5)

(*) indicates a disulfide bridge between the inercaptopropionic acid andthe cysteinamid.

550 ml of a methanol solution of 46.6 g ofMpr(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys (Trt)-NH₂ (SEQ ID NO: 5)(Mw=1374.8 g/mol, 20 mmol=1.00 equivalent) was treated with 130 g of awashed resin IRA 958 (Mw=1000 g/mol; 6.72 equivalents). After checkingthe exchange by HPLC, the resin was filtered and washed several timeswith 340 ml methanol. The combined organic phases were diluted by adding1500 ml methanol, 5400 ml dichloromethane and finally 180 ml water. 24 gof iodine was added to the diluted solution. After checking thecyclisation by HPLC, the remaining iodine is quenched by adding 700 mlof Na₂S₂O₃ (3.6% weight) in aqueous solution. The reaction mixture wasthen neutralized by 190 ml of resin acetate (Mw=720 g/mol; 57equivalents). After filtration and washing of the resin by 210 mlmethanol and 1800 ml water, the organic layer was separated from theaqueous one. The peptide (SEQ ID NO: 5) was extracted with 21 g of NaTPB(Mw=342.2 g/mol; 3 equivalents) from the neutralized aqueous layer byadding 10000 ml dichloromethane. After separation and evaporation of theorganic layer, the residue was dissolved in methanol (900 ml) and thenconcentrated in vacuo in order to eliminate most of the remainingdichloromethane. The methanol solution was suspended into 2400 g of awashed resin IRA 958 (Mw=1000 g/mol; 12.1 equivalents). After checkingthe exchange by HPLC and washing the resin, the combined organic phaseswere diluted by adding 240 ml of acetic acid. This solution was furtherpurified by preparative HPLC.

EXAMPLE 8 Storage of Mpr(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ TPBSalt (SEQ ID NO: 5)

Solid Mpr(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ TPB salt (SEQ IDNO: 5) was obtained by precipitation in accordance with the processaccording to the invention. The product was stored for more than 1 yearat a temperature of 20-25° C. The solid TPB salt had remainedsubstantially stable after the storage.

SEQUENCE LISTING <110>   SOLVAY SA <120>  Use of a TPB salt for the separation of biomolecules <130>   S200918<160>   5 <170>   PatentIn version 3.3 <210>   1 <211>   12 <212>   PRT<213>   Artificial <220> <223>   Synthetic peptide <400>   1Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1         5          10<210>   2 <211>   4 <212>   PRT <213>   Artificial <220> <223>  Synthetic Peptide <400>   2 Trp Arg Arg Lys 1 <210>   3 <211>   7<212>   PRT <213>   Artificial <220> <223>   Synthetic Peptide <400>   3Trp Trp Pro Trp Arg Arg Lys 1        5 <210>   4 <211>   10 <212>   PRT<213>   Artificial <220> <223>   Synthetic Peptide <400> 4Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1        5          10 <210>   5<211>   7 <212>   PRT <213>   Artificial <220> <223>   Synthetic Peptide<220> <221>   MISC_FEATURE <222>   (1) . .. (1) <223>  Mercaptopropionic acid <220> <221>   MISC_FEATURE <222>  (2) . . . (2) <223>   Homoarginine <400>   5Xaa Xaa Gly Asp Trp Pro Cys 1        5

1-18. (canceled)
 19. A process for the separation of a biomolecule froma liquid medium, wherein the biomolecule: i) is selected from the groupconsisting of a peptide, a peptide derivative, an oligonucleotide, anoligonucleotide derivative, a polysaccharide, and combinations thereof;and ii) comprises at least one cationic group; and wherein the processcomprises the steps of: a) providing a first organic solution containingthe biomolecule; and b) adding a tetraphenylborate (TPB) salt togetherwith an alkaline agent to the organic solution to form a biomolecule-TPBsalt in the first organic solution.
 20. The process of claim 19 whereinthe first organic solution containing the biomolecule is washed with anaqueous solution before the addition of the tetraphenylborate salt. 21.The process of claim 19, further comprising c) washing the first organicsolution containing the biomolecule-TPB salt with an aqueous solutionafter the addition of tetraphenylborate salt.
 22. The process of claim19, further comprising d) concentrating the first organic solutioncontaining the biomolecule-TPB salt.
 23. The process of claim 19,further comprising a step of e) adding to the first organic solutioncontaining the biomolecule-TPB salt a further organic solvent selectedfrom the group consisting of alcohols, amide-type solvents, halocarbons,esters, ethers, and combinations thereof, to form a second organicsolution containing the biomolecule-TPB salt.
 24. The process of claim22, further comprising a step of e) adding to the first organic solutioncontaining the biomolecule-TPB salt a further organic solvent selectedfrom the group consisting of alcohols, amide-type solvents, halocarbons,esters, ethers, and combinations thereof, to form a second organicsolution containing the biomolecule-TPB salt.
 25. The process accordingto claim 24, wherein the biomolecule is precipitated or crystallized asa solid biomolecule-TPB salt from the second organic solution.
 26. Theprocess of claim 22 comprising the step of performing a furthersynthesis step without isolation of the biomolecule-TPB salt, whereinthe said further synthesis step comprises coupling, deprotection andpurification.
 27. The process of claim 23 comprising the step ofperforming a further synthesis step without isolation of thebiomolecule-TPB salt, wherein the said further synthesis step comprisescoupling, deprotection and purification.
 28. The process of claim 19,wherein the tetraphenylborate salt is NaTPB.
 29. The process of claim19, wherein the at least one cationic group is an amino group or aguanidine group.
 30. The process of claim 19, wherein the biomoleculecomprises at least one guanidine group.